Continuous production of biodiesel fuel by enzymatic method

ABSTRACT

A method for continuously producing a fatty acid ester of the present invention comprises (a) mixing and agitating an oil and fat starting material and a lower alcohol, and supplying a mixture to one of the catalyst reaction tubes filled with a lipase; (b) producing a fatty acid ester and glycerin in the catalyst reaction tube; (c) introducing an outflowing liquid from the catalyst reaction tube into a glycerin separation tank, thereby collecting the glycerin; (d) adding a lower alcohol to a separated liquid obtained by separating the glycerin from the outflowing liquid, mixing and agitating an obtained material, and supplying a mixture to a following catalyst reaction tube; (e) repeating the steps (b) to (d) until supply to a last catalyst reaction tube is performed; and (f) collecting a fatty acid ester from the separated liquid obtained from the last catalyst reaction tube. According to the method of the present invention, the concentration of a lower alcohol can be strictly controlled and by product glycerin can be automatically removed.

TECHNICAL FIELD

The present invention relates to a method for continuously producing afatty acid ester useful as a biodiesel fuel using an enzymatic method,and an apparatus for the same.

BACKGROUND ART

In general, fossil fuels typified by petroleum and light oil are used asfuels for automobiles. These fossil fuels, especially, light oil usedfor diesel automobiles, contain a large amount of nitrogen compound andsulfur compound, so that a large amount of gas such as CO₂, NOx, SOx isexhausted from automobiles such as diesel automobiles. Since theseexhaust gases cause global warming and environmental pollution,reduction of the exhaust amount is an issue to be solved urgently.

As an alternative fuel to fossil fuels such as light oil, there are highexpectations in so-called biodiesel fuel, which uses oils and fatsproduced by naturally-occurring plants, animals, fishes, ormicroorganisms. Among these oils and fats, those used for food producingare often dumped into the environment and cause environmental problems.Therefore, expectations in biodiesel fuel made from a waste oil areparticularly high in view of prevention of air pollution and effectiveutilization of a waste oil.

Fatty acid esters obtained by transesterification between an oil and fatand a lower alcohol are preferably used as biodiesel fuels. A variety ofresearches have been conducted on an enzyme-catalytic method using alipase, as one method for producing a fatty acid ester (InternationalPublications WO 01/038553 and WO 00/12743). This production method hasmany advantages, for example, in that aftertreatment of by-productglycerin is easy, in that mild reaction conditions can be applied, andin that a free fatty acid in a starting material can be esterified (H.Fukuda et al., Journal of Bioscience and Bioengineering, 2001, Vol. 92,pp. 405-416).

Regarding production of a fatty acid ester using a lipase, researcheshave been intensively conducted on a batch-type transesterification inwhich an enzyme, an oil and fat, and a lower alcohol are agitated andmixed in a screw cap bottle or a reaction tank (Y. Shimada at al.,Journal of the American Oil Chemists' Society; 1999, Vol. 76, pp.789-793, and E. Y. Park et at., Bioresource Technology, 2008, Vol. 99,No. 8, pp. 3130-3135). In this method, it is necessary to be careful ofphysical damage to the enzyme due to agitation of the reaction mixture.Furthermore, in order to collect a product after the reaction, it isnecessary to perform a procedure that separates a product, an enzyme,and a by-product into layers after stopping agitation and allowing thereaction mixture to stand.

On the other hand, there is a report on production of a fatty acid esterusing a packed-bed reactor of a tube filled with a lipase through whichan oil and fat and a lower alcohol pass (Y. Watanabe at al., Journal ofthe American Oil Chemists' Society, 2000, Vol. 77, pp. 355-360, and K.Nie et al., Journal of Molecular Catalysis B: Enzymatic, 2006, Vol. 43,pp. 142-147). In this case, the enzyme is fixed in the tube, and, thus,the degree of physical damage to the enzyme is low, and the operationcan be performed for a long period of time. Moreover, since the reactorcan be filled with a large amount of enzyme, the method has a featurethat there is a significant increase in the amount of target substanceproduced per reactor unit volume and per reaction time. In researchesusing a packed-bed reactor, typical examples of which are shown by Y.Watanabe et al. (Journal of the American Oil Chemists' Society, 2000,Vol. 77, pp. 855-360) and K Nie et al. (Journal of Molecular CatalysisB: Enzymatic, 2006, Vol. 43, pp. 142-147), a method is applied in whichan oil and fat and a lower alcohol are supplied from an upper portion ofa reaction tube, a reaction mixture that has flown out of a lowerportion thereof is temporarily allowed to stand so as to be separatedinto layers, and then a fatty acid ester in an upper layer (alsocontaining unreacted oil and fat) is collected.

In general, a lower alcohol inhibits the activity of a lipase, and thusit is necessary to strictly control the ratio of lower alcohol containedin the reaction mixture. Furthermore, since the solubility of a loweralcohol in an oil and fat is extremely low, it is necessary to keep auniformly dispersed state such that a droplet of the alcohol is notformed in the oil and fat (Y. Shimada et al., Journal of MolecularCatalysis B: Enzymatic, 2002, Vol. 17, pp. 133-142). There is also amethod for reducing the alcohol inhibition by dissolving the reactionmixture in a hydrophobic organic solvent, such as hexane. However,according to the method the collection of the product is difficult, andthe production process is complicated.

Glycerin forms as a by-product during a procedure for producing a fattyacid ester. A certain amount of the glycerin accumulated forms a layeraround the enzyme. Since this layer of glycerin is hydrophilic, itgreatly affects the contact efficiency between unreacted oil and fat andthe enzyme. Moreover, part of a lower alcohol remaining in the reactionprocedure is dispersed in the glycerin layer, and locally increases theconcentration of alcohol near the enzyme, so that a lowered enzymaticactivity is caused (Y. Watanabe et al., Journal of the American OilChemists' Society, 2000, Vol. 77, pp. 855-860). Conventionally, there isa report on an attempt to remove glycerin by dialysis or by using anorganic solvent such as isopropanol (K. B. Bako et al., Biocatalysis andBiotransformation, 2002, Vol. 20, pp. 437-439, and Y. Xu et al.,Biocatalysis and Biotransformation, 2004, Vol. 22, pp. 45-48). However,there is a demand for a method for more easily removing glycerin, inview of industrialization of the process.

One aspect is that a method for continuously collecting a product for along period of time while supplying starting materials is desirable, anduse of a packed-bed reactor is advantageous for industrial production ofa biodiesel fuel using a lipase. However, another aspect is that it isnecessary to be careful of the possibility that a desirable yield offatty acid ester may not be obtained depending on the supply of an oiland fat and a lower alcohol to a reaction tube and the removalefficiency of by-product glycerin as described above. Therefore, it isnecessary to establish a method for continuously producing a biodieselfuel simultaneously considering these two aspects.

DISCLOSURE OF INVENTION

The object of the present invention is to provide a process capable ofstrictly controlling the concentration of a lower alcohol whileautomatically removing by-product glycerin in the production of a fattyacid ester from oils and fats using a lipase.

The present invention provides a method for continuously producing afatty acid ester in a reaction apparatus having a plurality of stages ofcatalyst reaction tubes filled with a lipase, comprising:

(a) mixing and agitating an oil and fat starting material and a loweralcohol, and supplying a mixture to one of the catalyst reaction tubes;

(b) producing a fatty acid ester and glycerin in the catalyst reactiontube to which the oil and fat starting material and the lower alcoholare supplied;

(c) introducing an outflowing liquid from the catalyst reaction tubeinto a glycerin separation tank, thereby collecting the glycerin;

(d) adding a lower alcohol to a separated liquid obtained by separatingthe glycerin from the outflowing liquid, mixing and agitating anobtained material, and supplying a mixture to a following catalystreaction tube;

(e) repeating the steps (b) to (d) until supply to a last catalystreaction tube is performed;

(f) introducing an outflowing liquid from the last catalyst reactiontube into a glycerin separation tank placed downstream of the lastcatalyst reaction tube, thereby collecting glycerin, and obtaining aseparated liquid obtained by separating the glycerin from the outflowingliquid; and

(g) collecting a fatty acid ester from the separated liquid obtained inthe step (f).

In an embodiment, a liquid flow rate in the reaction apparatus is atleast 2.15 cm/min.

In one embodiment, an amount of the lower alcohol supplied to each ofthe catalyst reaction tubes is 0.5 to 1.0 mol equivalent with respect tothe oil and fat starting material.

In a further embodiment, the number of stages of the catalyst reactiontubes is 2 to 10.

In an embodiment, the oil and fat starting material is a vegetable oiland fat, an animal oil and fat, a fish oil, an oil and fat produced by amicroorganism, a mixture thereof, or a waste oil thereof.

In one embodiment, the lower alcohol is methanol, ethanol, n-propanol,or n-butanol.

In a further embodiment, the method further comprising following thestep (f): (f) repeating the steps (a) to (f) using the separated liquidobtained in the step (f) as the oil and fat starting material.

The present invention also provides an apparatus for continuouslyproducing a fatty acid ester, comprising:

a plurality of stages of catalyst reaction tubes that are filled with alipase

a glycerin separation tank that is placed downstream of each of thecatalyst reaction tubes, and that separates an outflowing liquid fromthe catalyst reaction tube into glycerin and a separated liquid;

a lower alcohol supply port that is placed upstream of each of thecatalyst reaction tubes; and

a mixing means that is placed between each of the lower alcohol supplyports and each of the catalyst reaction tubes for mixing an oil and fatstarting material or the separated liquid and a lower alcohol;

wherein, in each stage of the catalyst reaction tubes, a mixture of theoil and fat starting material or the separated liquid from the glycerinseparation tank and the lower alcohol is supplied from an upper portionof the catalyst reaction tube, and an outflowing liquid from a lowerportion of the catalyst reaction tube is introduced into the glycerinseparation tank.

In an embodiment, a liquid flow rate in the apparatus is adjusted to atleast 2.15 cm/min.

In one embodiment, an amount of the lower alcohol supplied to each ofthe catalyst reaction tubes is adjusted to 0.5 to 1.0 mol equivalentwith respect to the oil and fat starting material.

In a further embodiment, the number of stages of the catalyst reactiontubes is 2 to 10.

According to the present invention, in transesterification between anoil and fat and a lower alcohol using a lipase as a catalyst, it ispossible to efficiently and continuously produce a fatty acid esterwhile automatically removing by-product glycerin. With the apparatus ofthe present invention, it is possible to efficiently and continuouslyperform a series of steps ranging from supply of starting materials tocollection of a fatty acid ester.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram showing the configuration of a reactionapparatus (packed-bed reactor) 100 of the present invention and the flowof continuous production of a biodiesel fuel using the same.

FIG. 2 is a schematic diagram showing a manner in which a fatty acidester and glycerin are separated in a glycerin separation tank 40.

FIG. 3 is a graph showing the concentration of fatty acid ester in aliquid flowing out of each stage of a catalyst reaction tube 10 at avolume flow rate of 250 to 1080 ml/h (a liquid flow rate of 2.15 to 9.30cm/min).

FIG. 4 is a graph showing the weight of glycerin separated in theglycerin separation tank 40 placed downstream of each stage of thecatalyst reaction tube 10.

FIG. 5 is a graph showing the concentration of methyl ester and theoutflow amount of glycerin in the case where 0.33 mol equivalents ofmethanol with respect to an oil and fat starting material are mixed witha liquid that is to be supplied to each stage of the catalyst reactiontube 10, and the mixture liquid is caused to pass through the tube at avolume flow rate of 540 ml/h (a liquid flow rate of 4.65 cm/min).

FIG. 6 is a graph showing the concentration of methyl ester in eachstage in the case where a methanolysis reaction is repeatedly performedusing two stages of catalyst reaction tubes.

BEST MODE FOR CARRYING OUT THE INVENTION Lipase

In the present invention, a lipase refers to an enzyme that has anability to degrade glyceride (also referred to as acylglycerol) intoglycerin or a partial glyceride and a fatty acid, and has an ability toproduce a fatty acid ester through transesterification in the presenceof a linear lower alcohol.

The lipase used in the present invention may be 1,3-specific ornonspecific. In view of production of a linear lower alcohol ester of afatty acid, a nonspecific lipase is preferable. Examples of the lipaseinclude: lipases derived from filamentous fungi belonging to GenusRhizomucor (Rhizomucor miehei), Mucor, Aspergillus, Rhizopus,Penicillium, and the like; lipases derived from yeasts belonging toGenus Candida (Candida antarctica, Candida rugosa, and Candidacylindracea), Pichia, and the like; lipases derived from bacteriabelonging to Genus Pseudomonas, Serratia, and the like; and lipasesderived from animals, such as hog pancreas. Commercially availablelipases are also used. Examples thereof include lipases derived fromRhizomucor miehei (Lipozyme IM60: manufactured by Novo Nordisk), lipasesderived from Candida antarctica (Novozyme 435: manufactured byNovozymes), lipases derived from Rhizopus delemar (Talipase:manufactured by Tanabe Seiyaku Co., Ltd.), lipases of Candida rugosa(Lipase OF: manufactured by Meito Sangyo Co., Ltd.), and lipases of thegenus Pseudomonas (Lipase PS and Lipase AK: manufactured by Amano EnzymeInc.).

In the present invention, an immobilized lipase refers to a lipase thatis immobilized on a given carrier. It is possible to use an immobilizedenzyme that is immobilized on a commonly used carrier, such as a resin,or to use a cell that produces and retains the lipase. Furthermore, asdescribed later, the cell may be further immobilized on a given carrier.Furthermore, it is effective to use mutually different types ofimmobilized lipases in each of the catalyst reaction tubes.

As the lipase that is immobilized on a carrier, ordinarily, a purifiedenzyme or a partially purified enzyme isolated or extracted from anatural product or a recombinant is used. Examples of the carrier onwhich a purified enzyme or a partially purified enzyme is immobilizedinclude carriers usually used for immobilizing an enzyme. Examplesthereof include organic high molecular compounds, such as variousion-exchange resins, and inorganic porous materials, such as ceramics.The immobilization is performed, for example, by applying methodsusually used by those skilled in the art, such as a carrier-bindingmethod, a crosslinking method, an entrapment method, and the like. Thecarrier binding method includes a chemical adsorption method or aphysical adsorption method comprising adsorbing to an ion-exchangeresin.

In the present invention, the cell that produces and retains the lipaseis bacteria, fungi, plant cells, or the like, but there is no limitationthereto. It is preferable to use yeasts and filamentous fungi. It isalso possible to use recombinants into which various lipase genes havebeen introduced.

The lipase-producing cell used in the present invention may beimmobilized on a carrier. As the material of the carrier that can beused in the present invention, foams or resins such as polyvinylalcohol, a polyurethane foam, a polystyrene foam, polyacrylamide, apolyvinyl formal resin porous material, a silicon foam, a celluloseporous material, or the like are preferable. It is preferable to use aporous carrier, for example, in view of shedding of cells with reducedgrowth and activity or dead cells. The size of the opening of the porousmaterial varies depending on the type of cells. The size enough forcells to enter and grow therein is suitable. A size of 50 μm to 1000 μmis preferable, but the size is not limited thereto. There is nolimitation regarding the shape of the carrier. In view of the strengthof the carrier, the cultivation efficiency or the like, spherical orcubical shapes are preferable. A preferable size is 1 mm to 50 mm indiameter for a spherical carrier, and 2 mm to 50 mm in length of theside for a cubical carrier.

Starting Materials for Fatty Acid Ester

Starting materials for the fatty acid ester are an oil and fat and alower alcohol.

As the oil and fat starting material, vegetable oils and fats, animaloils and fats, fish oils, oils and fats produced by microorganisms,mixtures thereof, or waste oils thereof are used preferbly. Examples ofvegetable oils and fats include soybean oil, rape seed oil, palm oil,olive oil, and the like. Examples of animal oils and fats include beeftallow, lard, sperm oil, mutton tallow, and the like. Examples of fishoils include sardine oil, tuna oil, squid oil, and the like. Examples ofoils and fats produced by microorganisms include oils and fats producedby microorganisms belonging to Genus Mortierella, Schizochytrium, andthe like. The waste oils refer to used plant and animal oils and fats,and examples thereof include Tempura (battered and deep fried foods)waste oils, and the like. Since the waste oils were heated to a hightemperature, they also contain hydrogenated, oxidized, or peroxidizedoils. These oils also can be used as the starting material. The oils maycontain water. Alternatively, the reaction mixture in which these oiland fat starting materials have once been treated with the lipase alsocan be used as the starting material.

A lower alcohol refers to an alcohol having 1 to 8 carbon atoms. Alinear lower alcohol is preferable, in particular, methanol, ethanol,n-propanol, or n-butanol is preferable.

Reaction Apparatus

In the present invention, a reaction apparatus refers to an apparatushaving a plurality of stages of connected catalyst reaction tubes thatare tubes, such as stainless steel tubes, filled with an immobilizedlipase or lipase-producing cells. FIG. 1 schematically shows theconfiguration of an embodiment of a reaction apparatus 100 as a typicalapparatus of the present invention, using, as an example, the case inwhich the reaction apparatus is a packed-bed reactor, but there is nolimitation thereto. This typical reaction apparatus 100 includes aplurality of stages of catalyst reaction tubes 10, glycerin separationtanks 40, lower alcohol supply ports, and a mixing means 50.

The catalyst reaction tubes 10 in FIG. 1 can be extended depending onthe reaction efficiency. The catalyst reaction tubes 10 preferably havea length of 1 to 2 m per stage, and an inner diameter of 1 to 5 cm, butthere is no limitation thereto. The number of stages of the catalystreaction tubes 10 is preferably 2 to 10, and more preferably 3 to 7, butthere is no limitation thereto. Furthermore, the catalyst reaction tubes10 are preferably made of a material selected in consideration of theviscosity of the oil and fat, and deterioration caused by the startingmaterials and the product. Examples of such a material include stainlesssteel and the like.

The catalyst reaction tubes 10 are filled with the lipase that isimmobilized by given means.

The catalyst reaction tubes 10 are connected to each other, for example,via tubes. The tubes connect the catalyst reaction tubes 10 such that anoil and fat starting material or a separated liquid is supplied to eachcatalyst reaction tube 10 from above, the separated liquid beingobtained after passing through its preceding catalyst reaction tube 10and the glycerin separation tank 40, and such that an outflowing liquidflows out of the lower portion of the catalyst reaction tube 10. Also,the tubes are preferably made of a material selected in consideration ofthe viscosity of the oil and fat, and deterioration caused by thestarting materials and the product, and more preferably made of siliconor Teflon (registered trademark).

In this embodiment, pressure gauges 20 are arranged in order to measurethe pressure in the catalyst reaction tubes 10. It is preferable to usepressure gauges that can indicate the pressure up to 1 MPa.

In this embodiment, pumps 30 are arranged in order to supply thestarting materials (the oil and fat and the lower alcohol) and theseparated liquid to the catalyst reaction tubes 10, or in order tosupply the outflowing liquid to the glycerin separation tanks 40, at agiven pressure or rate. It is preferable to use metering pumps having amaximum ejection pressure of approximately 0.4 to 1.0 MPa inconsideration of a pressure loss in the catalyst reaction tubes 10.

The glycerin separation tanks 40 are placed between the catalystreaction tubes 10. There is no particular limitation regarding a methodfor separating glycerin. For example, as shown in FIG. 2, the glycerinseparation tank 40 preferably has a space (e.g., a given space in asight glass) for retaining the outflowing liquid from the catalystreaction tube 10 (containing a fatty acid ester, unreacted oil and fat,and glycerin) for a given time. When the outflowing liquid is retainedin the glycerin separation tank 40, a fatty acid ester and unreacted oiland fat, and glycerin that are contained in the outflowing liquid areseparated into layers. The separated liquid containing the fatty acidester and the unreacted oil and fat overflows and is supplied to thefollowing catalyst reaction tube 10. The glycerin present in the lowerlayer is discharged downward when an electromagnetic valve is opened andclosed after the elapse of a given time, and collected by a receivingunit. In this separation, as described later in detail, it is importantto adjust the liquid flow rate (or the volume flow rate) in the reactionapparatus 100.

In the apparatus 100 of the present invention, lower alcohol supplyports are placed upstream of the respective stages of catalyst reactiontubes 10, and the lower alcohol is added from these supply ports to theseparated liquid. Here, it is necessary to keep a uniformly dispersedstate such that a droplet of the alcohol is not formed in the oil andfat, because the lower alcohol inhibits the activity of the lipase, andbecause the solubility of the lower alcohol to the oil and fat isextremely low. Therefore, in order to sufficiently mix the lower alcoholsupplied immediately before each stage, and the oil and fat startingmaterial or the outflowing liquid, the mixing means 50 is placed betweenthe lower alcohol supply port and the following catalyst reaction tube.Examples of the mixing means 50 include fillers in the tube, and astationary mixer. More specifically, for example, placing fillers suchas beads in a tube for supplying the oil and fat or the separated liquidto the catalyst reaction tubes 10 can facilitate mixing of a mixture ofthe oil and fat and the lower alcohol passing through the tube.

Furthermore, a constant-temperature water circulation apparatus ispreferably placed around the catalyst reaction tubes 10. Theconstant-temperature water circulation apparatus preferably can keep thetemperature of the reaction apparatus 100, in particular, the catalystreaction tubes 10 at 25° C. to 45° C. at which the enzyme reactionoccurs in a more preferable manner. Alternatively, the entire reactionapparatus 100, or the mixing means 50 and the catalyst reaction tubes 10may be placed in a constant-temperature chamber.

Method for Producing Fatty Acid Ester

The present invention is directed to a method for continuously producinga fatty acid ester in a reaction apparatus having a plurality of stagesof catalyst reaction tubes filled with a lipase, comprising:

(a) mixing and agitating an oil and fat starting material and a loweralcohol, and supplying a mixture to one of the catalyst reaction tubes;

(b) producing a fatty acid ester and glycerin in the catalyst reactiontube to which the oil and fat starting material and the lower alcoholare supplied;

(c) introducing an outflowing liquid from the catalyst reaction tubeinto a glycerin separation tank, thereby collecting the glycerin;

(d) adding a lower alcohol to a separated liquid obtained by separatingthe glycerin from the outflowing liquid, mixing and agitating anobtained material, and supplying a mixture to a following catalystreaction tube;

(e) repeating the steps (b) to (d) until supply to a last catalystreaction tube is performed;

(f) introducing an outflowing liquid from the last catalyst reactiontube to a glycerin separation tank placed downstream of the lastcatalyst reaction tube, thereby collecting glycerin, and obtaining aseparated liquid obtained by separating the glycerin from the outflowingliquid; and

(g) collecting a fatty acid ester from the separated liquid obtained inthe step (f).

In the present invention, it is possible to efficiently and continuouslyproduce a fatty acid ester, for example, using the reaction apparatus100 as shown in FIG. 1, as the reaction apparatus having a plurality ofstages of catalyst reaction tubes filled with a lipase. That is to say,in the reaction apparatus 100 described above, an oil and fat startingmaterial and a lower alcohol are sufficiently agitated and supplied tothe catalyst reaction tube 10, a lipase is caused to act in the catalystreaction tube 10, thereby producing a fatty acid ester, the outflowingliquid from the catalyst reaction tube 10 is introduced into theglycerin separation tank 40, thereby glycerin being collected, and theseparated liquid together with a lower alcohol is further supplied tothe following catalyst reaction tube 10. By repeating these operations,a fatty acid ester can be collected from the separated liquid from thelast catalyst reaction tube.

Alternatively, in the case where the number of stages of catalystreaction tubes is small, the separated liquid from the last catalystreaction tube may be used as the oil and fat starting material, and thereaction in this reaction apparatus may be repeated several times. Forexample, by repeatedly causing a liquid to pass three times through areaction apparatus having three stages of catalyst reaction tubes, it ispossible to obtain a separated liquid containing a fatty acid ester atthe same concentration as that in a reaction apparatus having ninestages of catalyst reaction tubes.

In the reaction apparatus 100, it is important to adjust the liquid flowrate (or the volume flow rate) in the reaction apparatus 100. If theliquid flow rate is low, the separation efficiency of glycerin from theoutflowing liquid in the glycerin separation tanks 40 is poor. Theliquid flow rate is determined as appropriate depending to the type ofalcohol, the diameter and the number of stages of the catalyst reactiontubes 10, the type of the oil and fat starting material, and the like.In the present invention, the liquid flow rate is usually at least 2.15cm/min, preferably at least 4.65 cm/min, more preferably at least 6.03cm/min, still more preferably at least 6.90 cm/min, even more preferablyat least 7.76 cm/min, and most preferably at least 8.62 cm/min.

The amount and the rate of lower alcohol supplied are determineddepending to the type of alcohol, the number of stages of the catalystreaction tubes 10, the type of the oil and fat starting material, theflaw rate, and the like. The amount of lower alcohol supplied to eachstage is preferably kept at 0.5 to 1.0 mol equivalent with respect tothe oil and fat starting material. If the amount of lower alcoholsupplied is small, the productivity of methyl ester and the separationefficiency of glycerin are poor. On the other hand, if the amount oflower alcohol supplied is large, the activity of the lipase in thecatalyst reaction tubes 10 may be inhibited. Furthermore, regarding therate of lower alcohol supplied, for example, in the case where atriolein starting material is caused to pass through the catalystreaction tubes 10 at 1000 ml/h, the rate of methanol supplied ispreferably 19.9 to 39.9 ml/h. Causing the lower alcohol to pass throughthe mixing means 50, such as fillers in the tube, a stationary mixer, orthe like can facilitate mixing of the lower alcohol and the oil and fat.

Transesterification between the oil and fat and the lower alcoholcatalyzed by the lipase is performed generally at 5° C. to 80° C.,preferably at 15° C. to 50° C., and more preferably at 25° C. to 45° C.The reaction temperature may be determined depending on a microorganismor an enzyme used. For example, in the case where a heat-resistantmicroorganism or enzyme is used, the reaction can be performed at arelatively high temperature.

The fatty acid ester after the reaction is separated and collected fromthe reaction mixture containing unreacted glyceride and lower alcoholthrough a separating operation usually used by those skilled in the art,such as distillation. The thus collected fatty acid ester can be used asa biodiesel fuel.

Examples Example 1 Reaction Apparatus

FIG. 1 shows a schematic diagram showing the configuration of thepacked-bed reactor 100 used in Examples. Stainless steel pipes (length 1m, inner diameter 15.7 mm, and volume 193.6 ml) were used as thecatalyst reaction tubes 10. These stainless steel pipes were filled withNovozyme 435 (manufactured by Novozymes) to an enzyme filing ratio of60% (v/v) to give the catalyst reaction tubes 10. The catalyst reactiontubes 10 were kept at 30° C., and the pressure gauges 20 were placedabove the tubes to confirm a pressure loss. The oil and fat startingmaterial was supplied to the upper portion of the catalyst reaction tube10 using the metering pump 30, end fillers were placed inside a supplytube for the catalyst reaction tube 10, thus facilitating mixing of theoil and fat and the lower alcohol.

Furthermore, the glycerin separation tanks 40 were placed under thecatalyst reaction tubes 10, and glycerin formed as a by-product duringthe reaction procedure was collected in each stage. As shown in FIG. 2,in the glycerin separation tank 40, the fatty acid ester, the unreactedoil and fat, and the glycerin contained in the outflowing liquid fromthe catalyst reaction tube 10 were retained in a given space in a sightglass, and the separated liquid (containing the fatty acid ester and theunreacted oil and fat) obtained by the separation performed therein anda newly added lower alcohol were supplied to the following catalystreaction tube 10 while being sufficiently mixed by the mixing means 50.

Example 2 Transesterification

First, 500 g of refined vegetable oil (Shirashime oil) was used as theoil and fat starting material, 9.07 g of methanol (0.5 mol equivalentswith respect to the oil and fat) was added to each stage, and themixture was caused to pass through the catalyst reaction tube 10. Here,the volume flow rate at the catalyst reaction tubes 10 was set to 250ml/h, 640 ml/h, or 1080 ml/h. These volume flow rates at the catalystreaction tubes 10 respectively correspond to liquid flow rates of 2.15cm/min, 4.65 cm/min, and 9.30 cm/min. After the oil and fat startingmaterial was caused to pass, the valves in the lower portions of theglycerin separation tanks 40 were opened, and the weight of by-productglycerin formed in each stage was measured. Then, 200 μl of theseparated liquid flowing out in each stage was collected, and theconcentration (content) of fatty acid methyl ester was analyzed.

The concentration (content) of fatty acid methyl ester (wt %) wasdetermined by a gas chromatography analysis using tricaprylin as theinternal standard. The analysis conditions were as follows.

Column: ZB-5HP (manufactured by Phenomenex, inner diameter 0.25 mm,length 15 m)

Column temperature:

-   -   Initial: 130° C., 2 min    -   Temperature rise: 350° C., 10° C./min        -   380° C., 7° C./min    -   Final temperature: 380° C., 10 min.

Injector temperature: 320° C.

Detector temperature: 380° C.

Carrier gas: helium gas (1.76)

Split ratio: 1/50

FIG. 3 shows the content of fatty acid methyl ester in the separatedliquid obtained in each stage. As the number of stages increased, thecontent of methyl ester in the outflowing liquid increased, and theconcentrations in the ninth stage were respectively 93.3 wt % (volumeflow rate 250 ml/h), 90.6 wt % (540 ml/h), and 84.8 wt % (1080 ml/h),and those in the 10th stage were respectively 95.3 wt % (540 ml/h) and88.9 wt % (1080 ml/h). The concentration of methyl ester in theseparated liquid obtained in each stage was affected by a change in thevolume flow rate, in other words, the retention time in the catalystreaction tubes 10, but it was shown that a sufficiently highconcentration of methyl ester was obtained even with a relatively shortretention time (approximately 10.8 min per stage at 1080 ml/h).

FIG. 4 shows the weight (cumulative amount) of glycerin collected by theseparation tank 40 in each stage. In the case where 500 g of oil and fatis completely converted to a fatty acid methyl ester, the total amountof glycerin formed as a by-product is approximately 52.04 g. In the casewhere the volume flow rate was 250 ml/h, glycerin was not practicallycollected even in the sixth stage where the content of methyl ester wasgreater than 68 wt %. This means that since the flow rate of theoutflowing liquid was low, a glycerin layer was retained near the enzymein the catalyst reaction tubes 10. In the case where the volume flowrate was 540 ml/h, the weight of glycerin separated increased as thenumber of stages increased, and 46.8 g of glycerin was separated instages up to the 10th catalyst reaction tube 10 where the content ofmethyl ester reached 95.3 wt %. This figure corresponds to 89.9% of theamount of glycerin that can be theoretically separated. Furthermore, inthe case where the volume flow rate was 1080 ml/h, 51.75 g of glycerin(99.4% of the theoretical amount) was separated in stages up to the 10thcatalyst reaction tube 10.

Example 3 Investigation on Reaction Conditions

Table 1 lists the number of stages in the packed-bed reactor used inthis Example, the concentration of fatty acid ester after the reactionin 10 stages, the productivity per unit reaction time and per unitreactor volume, the weight of glycerin separated, and the ratio of theglycerin amount with respect to the theoretical amount.

TABLE 1 Weight of Ratio of glycerin Volume Liquid Content of glycerinamount with respect flow rate flow rate Number methyl ester Productivityseparated to theoretical amount (ml/h) (cm/min) of stages (wt %) (g/h/L)(g) (%) 250 2.15 10 95.0 106.7 31.5 60.5 540 4.65 10 95.3 231.3 46.889.9 1080 9.30 10 88.9 431.4 51.8 99.4

It was shown that, with the same conditions as in Example 2, in the casewhere the oil and fat starting material or the separated liquid wascaused to pass through the catalyst reaction tubes at a volume flow rateof 540 ml/h or more, that is, at a liquid flow rate of 4.65 cm/min ormore, glycerin corresponding to approximately 90% of the theoreticalamount was separated, and that, in the case where the volume flow ratewas 1080 ml/h (the liquid flow rate was 9.30 cm/min), glycerincorresponding to 99% or more of the theoretical amount was separated. Inthe case where 10 stages of catalyst reaction tubes were taken as onereactor, the productivity of methyl ester per unit reaction time and perunit reactor volume was 106.7 g/h/L (250 ml/h), 231.3 g/h/L (540 ml/h),and 431.4 g/h/L (1.080 ml/h). As described by Y. Shimoda et al, (Journalof Molecular Catalysis B: Enzymatic, 2002, Vol. 17, pp. 133-142), in thecase where an oil and fat in an equal volume to that of the reactor istreated under batch-type reaction conditions using the same enzyme, anda product having a final concentration of methyl ester of 97.3 wt % isobtained after a reaction time of 48 hours, the productivity isestimated as 17.6 g/h/L. Therefore, it is shown that the process forproducing the fatty acid ester in the present invention is extremelyadvantageous in view of separation of the product and the by-product,and the productivity of the fatty acid ester per unit reaction time andper unit reactor volume.

Next, 0.33 mol equivalents of methanol with respect to the oil and fatwere mixed therewith in each stage, and the mixtures was caused to passthrough the stage at a volume flow rate of 540 ml/h. FIG. 5 shows thecontent of methyl ester and the outflow amount of glycerin (cumulativeamount) in this case. Under these conditions, the productivity of methylester and the separation efficiency of glycerin in each stage wereinferior to those in the case where 0.5 mol equivalents of methanol withrespect to the oil and fat starting material were mixed therewith.Accordingly, it is shown that adjustment of the amount of methanolsupplied to the catalyst reaction tubes significantly affects theapparatus ability.

Example 4 Investigation on Continuous Production using Two Stages ofCatalyst Reaction Tubes

In this Example, the number of stages of the catalyst reaction tubes 10was set to two in the packed-bed reactor 100 shown in FIG. 1. First, 0.5mol equivalents of methanol with respect to the oil and fat were mixedtherewith before each of a first and a second catalyst reaction tube 10,and the oil and fat was caused to pass through the tubes at a volumeflow rate of 640 ml/h (first cycle). Glycerin was separated from the oiland fat flowing out of the second catalyst reaction tube 10, 0.5 molequivalents of methanol with respect to the oil and fat were mixedtherewith, and then the oil and fat was again caused to pass through thefirst catalyst reaction tube 10 as in the first cycle (second cycle).The same operation was repeated two more times. FIG. 6 shows the contentof fatty acid methyl ester in the separated liquid obtained in eachstage.

FIG. 6 clearly shows that, in the case where a methanolysis reaction wasrepeated four times in the two stages of catalyst reaction tubes, afatty acid ester having a high purity was obtained. It is shown that, inthe case where a liquid is continuously and repeatedly caused to passthrough a reactor having two stages of catalyst reaction tubes in thismanner, a fatty acid ester having a high purity can be obtained as inthe case where a reactor having many stages of catalyst reaction tubesis used. Therefore, it is shown that the cost of the apparatus can bereduced by reducing the number of stages of catalyst reaction tubes.

INDUSTRIAL APPLICABILITY

According to the method of the present invention, a fatty acid ester canbe produced through a continuous procedure of adding a lower alcoholwithout stopping the reaction, and automatically separating by-productglycerin, and thus, production efficiency is high, and production costcan be reduced. Furthermore, the separated glycerin does not requirespecial washing treatment, and thus, the environmental load is low, andthe produced fatty acid ester is provided as a biodiesel fuel with lessenvironmental pollution.

1. A method for continuously producing a fatty acid ester in a reactionapparatus having a plurality of stages of catalyst reaction tubes filledwith a lipase, comprising: (a) mixing and agitating an oil and fatstarting material and a lower alcohol, and supplying a mixture to one ofthe catalyst reaction tubes; (b) producing a fatty acid ester andglycerin in the catalyst reaction tube to which the oil and fat startingmaterial and the lower alcohol are supplied; (c) introducing anoutflowing liquid from the catalyst reaction tube into a glycerinseparation tank, thereby collecting the glycerin; (d) adding a loweralcohol to a separated liquid obtained by separating the glycerin fromthe outflowing liquid, mixing and agitating an obtained material, andsupplying a mixture to a following catalyst reaction tube; (e) repeatingthe steps (b) to (d) until supply to a last catalyst reaction tube isperformed; (f) introducing an outflowing liquid from the last catalystreaction tube into a glycerin separation tank placed downstream of thelast catalyst reaction tube, thereby collecting glycerin, and obtaininga separated liquid obtained by separating the glycerin from theoutflowing liquid; and (g) collecting a fatty acid ester from theseparated liquid obtained in the step (f).
 2. A method according toclaim 1, wherein a liquid flow rate in the reaction apparatus is atleast 2.15 cm/min.
 3. A method according to claim 1, wherein an amountof the lower alcohol supplied to each of the catalyst reaction tubes is0.5 to 1.0 mol equivalent with respect to the oil and fat startingmaterial.
 4. A method according to claim 1, wherein the number of stagesof the catalyst reaction tubes is 2 to
 10. 5. A method according toclaim 1, wherein the oil and fat starting material is a vegetable oiland fat, an animal oil and fat, a fish oil, an oil and fat produced by amicroorganism, a mixture thereof, or a waste oil thereof.
 6. A methodaccording to claim 1, wherein the lower alcohol is methanol, ethanol,n-propanol, or n-butanol.
 7. A method according to claim 1, furthercomprising following the step (f): (f′) repeating the steps (a) to (f)using the separated liquid obtained in the step (f) as the oil and fatstarting material.
 8. An apparatus for continuously producing a fattyacid ester, comprising: a plurality of stages of catalyst reaction tubesthat are filled with a lipase; a glycerin separation tank that is placeddownstream of each of the catalyst reaction tubes, and that separates anoutflowing liquid from the catalyst reaction tube into glycerin and aseparated liquid; a lower alcohol supply port that is placed upstream ofeach of the catalyst reaction tubes; and a mixing means that is placedbetween each of the lower alcohol supply ports and each of the catalystreaction tubes for mixing an oil and fat starting material or theseparated liquid and a lower alcohol; wherein, in each stage of thecatalyst reaction tubes, a mixture of the oil and fat starting materialor the separated liquid from the glycerin separation tank and the loweralcohol is supplied from an upper portion of the catalyst reaction tube,and an outflowing liquid from a lower portion of the catalyst reactiontube is introduced into the glycerin separation tank.
 9. An apparatusaccording to claim 8, wherein a liquid flow rate in the apparatus isadjusted to at least 2.15 cm/min.
 10. An apparatus according to claim 8,wherein an amount of the lower alcohol supplied to each of the catalystreaction tubes is adjusted to 0.5 to 1.0 mol equivalent with respect tothe oil and fat starting material.
 11. An apparatus according to claim8, wherein the number of stages of the catalyst reaction tubes is 2 to10.